Project Summary Pseudomonas aeruginosa (PA) and Klebsiella pneumoniae (Kp) are among the most common causes of health care associated pneumonia. Both are opportunistic pathogens, often highly resistant to antibiotics and form biofilms that thwart effective phagocytosis. As Gram-negative bacteria, both express LPS that induces TLR4/Trif mediated IFN signaling, a host response stimulated by ligation of intracellular receptors typically associated viral infection, yet also important in the eradication of extracelluar bacterial pathogens from the lung. Despite these similarities, each organism activates a distinctive host innate immune response: PA clearance is initiated by epithelial NF-?B signaling very shortly after infection, whereas Kp is able to replicate within the airway and effective clearance begins to occur days following infection. Both pathogens stimulate type III (IFN-?) signaling in the airway mucosa, a response that is detrimental to eradication of the infection. In the experiments detailed in this proposal, we will test the hypothesis that the relative induction f epithelial versus immune cell signaling by these organisms is an important factor in their successful eradication from the airway. We will establish the importance of IL-28R-dependent type III IFN signaling in the clearance of Kp and PA in several murine models of pneumonia including the humanized NSG mouse. Using Cxcr3-/- mice, we will examine the role of CXCR3 chemokines and the contribution of recruited T cells and NK cells in the pathogenesis of acute bacterial pneumonia, and will confirm the involvement of these chemokines in human infection. We postulate that the ability of PA and Kp to selectively activate NF-?B/MAPK signaling versus the IFN cascades is regulated by their relative ability to activate the host deubiquitinating enzymes CYLD and USP25.Induction of CYLD activity is also expected to be a key factor in pathogenesis, associated with lung pathology due to necroptosis, a proinflammatory mechanism of host cell death. Given the importance of both of these pathogens in health care settings, understanding their specific mechanisms of pathogenesis will be critical to develop adjunctive immunotherapy to prevent or to treat hospital associated pneumonia.